重组Anti-Histone H2A抗体[EPR17470] - ChIP Grade

• ChIC/CUT&RUN-seq

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ChIC/CUT&RUN sequencing - Anti-Histone H2A antibody [EPR17470] - ChIP Grade (AB177308)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2 x 10^5 HeLa (Human cervix adenocarcinoma epithelial cell line) cells and 2 µg of ab177308 [EPR17470]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown. Additional screenshots of mapped reads can be found in the Protocol booklet in the Product Protocol section. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2A antibody [EPR17470] - ChIP Grade (AB177308)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling Histone H2A using ab177308 at 1/1000 dilution. A Goat Anti-Rabbit IgG H&L (HRP) (ab97051) was used as secondary at 1/500 dilution. Counterstained with Hematoxylin. Nucleus staining on epithelial cells of Human colon tissue was observed.
Negative control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2A antibody [EPR17470] - ChIP Grade (AB177308)

Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling Histone H2A using ab177308 at 1/1000 dilution. A Goat Anti-Rabbit IgG H&L (HRP) (ab97051) was used as secondary at 1/500 dilution. Counterstained with Hematoxylin. Nucleus staining on epithelial cells of rat colon tissue was observed.
Negative control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2A antibody [EPR17470] - ChIP Grade (AB177308)

Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling Histone H2A using ab177308 at 1/1000 dilution. A Goat Anti-Rabbit IgG H&L (HRP) (ab97051) was used as secondary at 1/500 dilution. Counterstained with Hematoxylin. Nucleus staining on epithelial cells of mouse colon tissue was observed.
Negative control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution..

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Histone H2A antibody [EPR17470] - ChIP Grade (AB177308)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling Histone H2A with ab177308 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green).

Confocal image showing nuclear staining on HeLa cell line.

The nuclear counter stain is DAPI (blue).

Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows :
1. ab177308 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

• ChIP

Lab

ChIP - Anti-Histone H2A antibody [EPR17470] - ChIP Grade (AB177308)

Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25μg of chromatin, 2μg of ab177308 (blue), and 20μl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.

• WB

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Western blot - Anti-Histone H2A antibody [EPR17470] - ChIP Grade (AB177308)

5% NFDM/TBST : Blocking and diluting buffer.

All lanes:

Western blot - Anti-Histone H2A antibody [EPR17470] - ChIP Grade (ab177308) at 1/5000 dilution

All lanes:

HeLa (Human epithelial cells from cervix adenocarcinoma) cell lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 14 kDa

Observed band size: 14 kDa

false

Exposure time: 30s

• WB

Supplier Data

Western blot - Anti-Histone H2A antibody [EPR17470] - ChIP Grade (AB177308)

5% NFDM/TBST : Blocking and diluting buffer.

All lanes:

Western blot - Anti-Histone H2A antibody [EPR17470] - ChIP Grade (ab177308) at 1/1000 dilution

Lane 1:

mouse brain lysate at 10 µg

Lane 2:

mouse heart lysate at 10 µg

Lane 3:

mouse kidney lysate at 10 µg

Lane 4:

mouse spleen lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 14 kDa

Observed band size: 14 kDa

false

Exposure time: 1s

• PepArr

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Peptide Array - Anti-Histone H2A antibody [EPR17470] - ChIP Grade (AB177308)

ab177308 was tested in Peptide Array against 501 different modified and unmodified histone peptides; each peptide is printed on the array at six concentrations (each in triplicate).
Circle area represents affinity between the antibody and a peptide : all antigen-containing peptides are displayed as red circles, all other peptides as blue circles. The affinity is calculated as area under curve when antibody binding values are plotted against the corresponding peptide concentration. Each circle area is normalized to the peptide with the strongest affinity.
The complete dataset, including full list of all peptides and information on the position of each peptide in the diagram, is available under the Product Protocol section.

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-Histone H2A antibody [EPR17470] - ChIP Grade (AB177308)

CUT&RUN was performed using the ChIC/CUT&RUN pAG-MNAse ab285373, 2.5 x 10^5 HeLa cells and 2μg of ab177308 [EPR17470]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. Additional screenshots of mapped reads can be downloaded here.